heterogeneity of heat-resistant proteases from milk pseudomonas species

International Journal of Food Microbiology 133 (2009) 68–77

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International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r.com/ locate / i j foodmicro

Heterogeneity of heat-resistant proteases from milk Pseudomonas species

Sophie Marchand a,⁎, Gonzalez Vandriesche c, An Coorevits d,e, Katleen Coudijzer a, Valerie De Jonghe a,Koen Dewettinck b, Paul De Vos d, Bart Devreese c, Marc Heyndrickx a, Jan De Block a

a Institute for Agricultural and Fisheries Research - Technology and Food (ILVO-T&V), Brusselsesteenweg 370, 9090 Melle, Belgiumb Ghent University, Faculty of Bioscience Engineering, Department of Food Safety and Food Quality, Laboratory of Food Technology and Engineering, Coupure Links 653, 9000 Ghent, Belgiumc Ghent University, Faculty of Science, Department of Biochemistry, Physiology and Microbiology, Laboratory for Protein Biochemistry and Biomolecular Engineering, K.L. Ledeganckstraat 35,9000 Ghent, Belgiumd Ghent University, Faculty of Science, Department of Biochemistry, Physiology and Microbiology, Laboratory of Microbiology, K.L. Ledeganckstraat 35, 9000 Ghent, Belgiume University College Ghent, Faculty of Applied Engineering Sciences, Laboratory of Biochemistry and Brewing, Voskenslaan 270, 9000 Ghent, Belgium

⁎ Corresponding author. Tel.: +32 9 2723000; fax: +E-mail address: [email protected]

0168-1605/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.ijfoodmicro.2009.04.027

a b s t r a c t
a r t i c l e i n f o
Article history:Received 2 February 2009Received in revised form 27 April 2009Accepted 29 April 2009

Keywords:Pseudomonas spp.aprXMilk spoilageHeat stable proteases

Pseudomonas fragi, Pseudomonas lundensis andmembers of the Pseudomonas fluorescens groupmay spoil UltraHigh Temperature (UHT) treatedmilk and dairy products, due to the production of heat-stable proteases in thecold chain of rawmilk. Since the aprX gene codes for a heat-resistant protease in P. fluorescens, the presence ofthis genehas also been investigated in othermembers of the genus. For this purpose an aprX-screening PCR testhas been developed. Twenty-nine representatives of important milk Pseudomonas species and thirty-fivereference strains were screened. In 42 out of 55 investigated Pseudomonas strains, the aprX genewas detected,which proves the potential of the aprX-PCR test as a screening tool for potentially proteolytic Pseudomonasstrains in milk samples. An extensive study of the obtained aprX-sequences on the DNA and the amino acidlevel, however, revealed a large heterogeneity within the investigated milk isolates. Although this hete-rogeneity sets limitations to a general detectionmethod for all proteolytic Pseudomonas strains inmilk, it offersa great potential for the development of a multiplex PCR screening test targeting individual aprX-genes.Furthermore, our data illustrated the potential use of the aprX gene as a taxonomic marker, which may help inresolving the current taxonomic deadlock in the P. fluorescens group.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Bacterial spoilage still causes significant losses for the foodindustry. Psychrotrophic (or psychrotolerant) bacteria, predominantlyPseudomonas spp. (Craven andMacauley, 1992), may cause spoilage ofmilk and milk products, due to their production of heat stableextracellular enzymes (Driessen, 1983; Sorhaug and Stepaniak, 1997).Although pseudomonads are easily destroyed by the heating settingsapplied by the dairy industry, many of their enzymes survive heattreatments even in UHT-conditions and remain active in derived dairyproducts (Griffiths et al., 1981; Mitchell and Ewings, 1985; Sorhaugand Stepaniak, 1997; Chen et al., 2003). The presence of heat stableenzymes in UHT milk and dairy products may cause instabilityproblems and spoilage, long before the expiry date of the products isreached (Driessen, 1983). Heat stable lipases may be responsible forthe hydrolysis of fat, which leads to rancidity (Mencher et al., 1965;Griffiths et al., 1981; Fox and Stepaniak, 1983; Stead, 1986; Woodset al., 2001). The proteases are predominantly active towards the

32 9 2723001.e (S. Marchand).

ll rights reserved.

casein fraction, which causes gelling of UHT milk and/or the for-mation of bitter off-flavors (Mitchell and Marshall, 1989; Chen et al.,2003; Datta and Deeth, 2003). Since lipolytic spoilage is lessfrequently reported (Law, 1979; Champagne et al., 1994; Shah, 1994;Koka and Weimer, 2000), most research has focused on expressionand production of proteases (Barach et al., 1976; McKellar, 1989;Mitchell and Marshall, 1989; Kohlmann et al., 1991; Ching-hsing andMcCallus, 1998; Liao and McCallus, 1998; Koka and Weimer, 2000;Nicodème et al., 2005; Liu et al., 2007; Dufour et al., 2008). Thosethermostable proteases have many properties similar to the serralysinprotease family of which the AprX protein from Pseudomonasaeruginosa is the most extensively studied (Okuda et al., 1990;Duong et al., 1992; Baumann et al., 1993; Miyatake et al., 1995; Kimet al., 1997). The aprX gene, encoding an alkaline metalloprotease, isbelieved to be responsible for the spoilage of milk (Dufour et al.,2008). Although, this protease gene is widespread over numerousPseudomonas spp. (Duong et al., 1992; Liao and McCallus, 1998;Kawai et al., 1999; Kumeta et al., 1999; Chessa et al., 2000; Chabeaud etal., 2001), the production process is still not completely understoodand appears to be very complex. Quorum sensing (Juhas et al., 2005;Liu et al., 2007), temperature (McKellar and Cholette, 1987; Burger etal., 2000; Nicodème et al., 2005), iron content (McKellar, 1989; Woods

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69S. Marchand et al. / International Journal of Food Microbiology 133 (2009) 68–77

et al., 2001) and phase variation (Chabeaud et al., 2001; van denBroeck et al., 2005) regulate and influence the production process atdifferent levels. Pseudomonas fragi and Pseudomonas lundensis alongwith members of the Pseudomonas fluorescens group are most fre-quently involved in the spoilage of dairy products (Deeth et al., 2002;Ercolini et al., 2007; Marchand et al., 2009). Although the speciesP. fluorescens is very heterogeneous and exact identification remainscontroversial and difficult (Bossis et al., 2000; Ercolini et al., 2007;Marchand et al., 2009), most of the biochemical research on proteaseproperties has focused on this species in the past (Kim et al., 1997;Ching-hsing and McCallus, 1998; Kumeta et al., 1999). P. fluorescensproduces only one protease, typically an alkaline zinc metalloproteasewith a pH optimum of 6.5–8 (Fairbairn and Law, 1986; Woods et al.,2001). The alkaline metalloprotease (aprX) and lipase (lipA) genes ofP. fluorescens B52 are located at opposite ends of an operon thatcontains eight genes and spans 14 kb; the ‘aprX–lipA’ operon alsocontains a protease inhibitor, type I secretion functions and twoautotransporter proteins (Woods et al., 2001; McCarthy et al., 2004).The aprX gene products of two P. fluorescens strains revealed estimatedmolecular weights of 50 kDa (Ching-hsing and McCallus, 1998) and48.9 kDa (Kumura et al., 1999). Similar molecular weights werereported by other researchers, investigating food spoiling proteases(Noreau and Drapeau, 1979; Kohlmann et al., 1991; Schokker and vanBoekel, 1997; Koka and Weimer, 2000; Rajmohan et al., 2002;Nicodème et al., 2005). The family of serralysin proteases appears tobe highly conserved. Typical similarities in amino acid sequence areobserved: a zinc-binding motif (xxxQTLTHEIGHxxGLxxGLxHPx), acalcium binding domain characterized by the presence of four glycinerich repeats (GGxGxD), a high content of hydrophobic amino acidsand no cysteine residues (Rawlings and Klostermeyer, 1995; Kumetaet al., 1999; Kumura et al., 1999). The amino acid sequence of themetalloproteases of more distantly related bacteria, such as Serratiamarcescens and Erwinia chrysanthemi still share a homology percen-tage of 50 to 60% with the alkaline proteases of P. fluorescens andP. aeruginosa (Ching-hsing and McCallus, 1998; Kumeta et al., 1999;Kumura et al., 1999).

To prevent deterioration of dairy products, rapid detection ofPseudomonas strains with high protein degrading effects in milk isvery convenient. PCR methods based on the aprX gene might enhanceand accelerate this detection process. Yet, published methods possessrather low sensitivity, when applied directly to milk. It is also notalways clear which potential hazardous strains can be detected withthe proposed methods (Martins et al., 2005). More research is alsoneeded towards the proteolytic capacity of aprX-possessing strains inmilk. Evidently, possession implies not necessarily expression of thegene. Considering the complexity of the aprX gene expression/regu-lation, the development of an immunological detection method forthe protease proteins might also be a fruitful option. The developmentof such a method necessitates accurate insights into the nature andstructure of the milk spoiling proteases. Therefore the presence anddegree of conservation of the aprX gene was explored in the pre-dominant proteolytic psychrotrophic bacteria isolated from raw milk.Our goals were: i/detection of the aprX gene in the predominant milkspoilers, in particular P. lundensis, P. fragi and members of theP. fluorescens group, ii/assessing the variability within the producedmilk spoiling proteases at the nucleotide and the amino acid level andiii/construction of an aprX gene based molecular framework forclassification of proteolytic psychrotrophic milk spoiling bacteria.

2. Materials and methods

2.1. Bacterial strains

In a previous screening of Belgian raw milk samples (Marchandet al., 2009) a large set of proteolytic psychrotrophic bacteria wasisolated and identified by a polyphasic approach. From this collection,

29 strains, which produced the highest level of heat-resistantproteases, were selected as representatives (of 81 strains) to coverthe most encountered proteolytic psychrotrophic spoilers in rawmilk.More specifically, five P. lundensis (W22b, W52b, Z24a, Z35c andZ60a), nine P. fragi (W5b, W12d, W29a, W50b, W51a, W52d, Z41b,Z53a and Z58c) and fifteen members of the P. fluorescens group (W2a,W30a, W51e, W5a, Z22b, Z34b, W12b, W31b, W38a, Z34a, Z38b,W15a,W17a,W17c and Z57b) (strains could not be identified onto thespecies level due to the weak taxonomic species delineation in thisgroup of bacteria) were included. All milk strains produced heatresistant proteases, which survived heating at 95 °C for 8.45 min(residual activity ±75%) (Marchand et al., 2008, 2009). Referencestrains were obtained from the BCCM/LMG bacteria collection, GhentUniversity, Belgium. All strains used are listed in Table 1.

2.2. Assessment of proteolytic activity

All milk and reference strains were tested for their proteolyticactivity by agar diffusion assays at 7 °C for 10 days, at 22 °C for 7 daysand at 30 °C for 3 days. Proteolytic enzyme production was visualizedon milk agar. To prepare the milk medium, commercial UHT milk wasused. A 2%milk agar mediumwas prepared by adding 8ml sterile UHTmilk to 400 ml nutrient agar (Oxoid, Basingstoke, Hampshire,England) incubated at 45 °C for 45 min. The presence of a clear zonearound the colonies after incubation was indicative for proteolysis.

2.3. Casein zymography

To estimate the molecular mass of the bacterial proteases, a caseinzymography was performed. Strains were grown in sterile full fatUHT-milk during 2 days at 22 °C. Subsequently, milk samples wereheated at 95 °C for 8 min and 45 s as described by Marchand et al.(2009) to select for heat-stable proteases. After heating, milk sampleswere cooled down on ice and diluted thirty-three times in loadingbuffer (62.5 mM Tris, 25% glycerol, 0.01% (w/v) bromophenol blue, 4%(w/v) Sodium Dodecyl Sulfate (SDS), pH 6.8). 40 μl of 0.1% proteinsolutions were loaded for electrophoresis on a 12.5% zymogramCriterion™ Precast gel, containing 0.1% (w/v) casein (Biorad, NazarethEke, Belgium) under nonreducing conditions. The samples were run at150 V for 60 min. After electrophoresis, the gels were washed in 2.5%(v/v) Triton X-100 for 30 min and then incubated for 20 h at 37 °C indevelopment buffer (50 mM Tris, 200 mM NaCl, 5 mM CaCl2·2H2Oand 0.02% (v/v) Brij 35 (Sigma, Bornem, Belgium), pH 7.5). Afterincubation, the gel was stained with 0.1% (w/v) Coomassie BrilliantBlue dissolved in a mixture of 40% ethanol (v/v) and 10% acetic acid(v/v), followed by discoloration in a solution containing 40% ethanol(v/v) and 10% acetic acid (v/v) until proteolytic activity appeared asclear bands on a blue background.

2.4. Primer design, PCR development and sequencing of the proteasegene

The nucleotide sequences (AF083061, AB015053, AF004848,DQ146945 and AB013895) of the metalloprotease gene (aprX) fromvarious P. fluorescens strains were aligned. Four protease primers weredesigned based on conserved regions: SM1F (5′-TTG-CAA-ACA-AGG-AAG-TAC-GT-3′), SM2F (5′-AAA-TCG-ATA-GCT-TCA-GCC-AT-3′),SM3R (5′-TTG-AGG-TTG-ATC-TTC-TGG-TT-3′) and SM4R (5′-GTG-AAG-TCR-AAG-ATC-TTG-TC-3′). DNA for the PCR-reaction wasobtained from approximately 5 colonies by alkaline lysis of the cellsin 50 μl sodium hydroxide (0.1 M) and 50 μl SDS (0.25% w/v). Thebacterial lysatewas heated for 17min at 90 °C and immediately cooledon ice. PCR reaction was carried out in a final volume of 50 μlcontaining 10× PCR buffer with 1 U Taq DNA polymerase (Eurogentec,Seraing, Belgium), 1.5 mM MgCl2, 5% (v/v), Tween-20 0.1% (w/v)gelatine (Sigma, Bornem, Belgium), each of the deoxynucleoside

Table 1An overview of tested strains and their proteolytic characterization.

Proteolytic psychrotrophic pseudomonads isolatedfrom cooled Belgian raw milk samplesa

Incubation at 30 °Con milk agar



Detection of aprXgene with primerset SM2/SM3

Accessionnumber ofaprX sequenceGrowth Proteolysis Growth Proteolysis Growth Proteolysis

R-35697 Pseudomonas fragi W29a + − + + + + + FM872353R-35701 Pseudomonas fragi W5b + − + + + + + FM872354R-35703 Pseudomonas fragi W52d + − + + + + + FM872355R-35705 Pseudomonas fragi Z58c + − + + + + + FM872356R-35706 Pseudomonas fragi Z53a + − + + + + + FM872357R-35709 Pseudomonas fragi Z41b + − + + + + + FM872358R-35710 Pseudomonas fragi W50b + − + + + + + FM872359R-35717 Pseudomonas fragi W12d + − + + + + −R-35719 Pseudomonas fragi W51a + − + + + + + FM872360R-35702 Pseudomonas lundensis W52b + + + + + + −R-35711 Pseudomonas lundensis W22b + + + + + + −R-35721 Pseudomonas lundensis Z60a + + + + + + −R-35723 Pseudomonas lundensis Z24a + + + + + + −R-35724 Pseudomonas lundensis Z35c + + + + + + −R-35708 Pseudomonas fluorescens Z34b + + + + + + + FM872361R-35700 Pseudomonas fluorescens W2a + + + + + + + FM872362R-35704 Pseudomonas sp. Z22b + + + + + + + FM872363R-35712 Pseudomonas sp. W30a + + + + + + + FM872364R-35698 Pseudomonas sp. W15a + + + + + + + FM872365R-35722 Pseudomonas sp. Z57b + + + + + + + FM872366R-35713 Pseudomonas sp.W12b + + + + + + + FM872367R-35716 Pseudomonas sp. W38a + + + + + + + FM872368R-35699 Pseudomonas sp. W31b + + + + + + + FM872369R-35720 Pseudomonas fluorescens W51e + + + + + + + FM872370R-35707 Pseudomonas fluorescens Z34a + + + + + + + FM872371R-35725 Pseudomonas fluorescens Z38b + + + + + + + FM872372R-35715 Pseudomonas sp. W17a + + + + + + + FM872373R-35718 Pseudomonas sp. W17c + + + + + + + FM872374R-35714 Pseudomonas sp. W5a + + + + + + −

Reference strainsb

LMG 5825 Pseudomonas fluorescens biotype A + − + + + + + FM872375LMG 5830 Pseudomonas fluorescens biotype A + + + + + + + FM872376LMG 1794 T Pseudomonas fluorescens biotype A + − + + + + + FM872377LMG 6812 Pseudomonas fluorescens + − + + + + + FM872378LMG 17764T Pseudomonas rhodesiae + − + + + − + FM872379LMG 2342T Pseudomonas tolaasii + + + + + + + FM872380LMG 14674 Pseudomonas fluorescens biotype C + + + + + + + FM872381LMG 5822 Pseudomonas fluorescens biotype C + − + + + + + FM872382LMG 1244 Pseudomonas fluorescens biotype C + − + + + + + FM872383LMG 5938 Pseudomonas fluorescens biotype C + − + + + − + FM872384LMG 1245T Pseudomonas chlororaphis subsp. aureofaciens + + + + + + + FM872385LMG 5004T Pseudomonas chlororaphis subsp. chlororaphis + + + + + + + FM872386LMG 5167 Pseudomonas fluorescens biotype G + + + + + + + FM872387LMG 2191T Pseudomonas fragi + − + − + − + FM872388LMG 5919 Pseudomonas fragi + − + − + − + FM872389LMG 5920 Pseudomonas fragi + − + − + − + FM872390LMG 5940 Pseudomonas fluorescens biotype G + + + + + + + FM872391LMG 21611T Pseudomonas azotoformans + + + + + + + FM872392LMG 5168 Pseudomonas fluorescens biotype F + − + − + − + FM872393LMG 21604T Pseudomonas gessardii + − + + + − + FM872394LMG 13517T Pseudomonas lundensis + − + − + − −LMG 14572 Pseudomonas marginalis + − + − + − −LMG 1247T Pseudomonas syringae + + + + + − −LMG 1242T Pseudomonas aeruginosa + + + + + − −LMG 7041T Pseudomonas luteola + − + − − − −LMG 24276T Pseudomonas psychrophila + − + − + − −LMG 8337T Chryseobacterium indologenes + − + − + − −LMG 6923T Bacillus cereus + + + − - − −LMG 984 Acinetobacter baumanii + + + + - − −LMG 2792T Serratia marcescens + + + − - − −LMG 2844T Aeromonas hydrophila + + + + + − −LMG 2092T Escherichia coli + − + − − − −LMG 2468T Dickeya chrysthemi + − + − − − −LMG 1222T Burkholderia cepacia + + + + − − −LMG 8347T Sphingobacterium spiritivorum + − + − − − −

a All strains were isolated from Belgian raw milk samples and identified by a polyphasic approach (Marchand et al., 2009). R-numbers represent research collection numbers asused by BCCM/LMG Bacteria Collection, Ghent University, Belgium.

b All reference strains were obtained from the BCCM/LMG Bacteria Collection, Ghent University, Belgium.

70 S. Marchand et al. / International Journal of Food Microbiology 133 (2009) 68–77


triphosphates at a concentration of 200 μM, each of the primers at aconcentration of 1 μMand 2 μl of template DNA. PCR amplificationwasperformed with a Perkin-Elmer thermocycler 9700 (Applied Biosys-tems, Foster City, CA, USA). Different primer combinations weretested. PCR amplification was performed for the four sets of primers(SM1F/SM3R, SM2F/SM3R, SM1F/SM4R and SM2F/SM4R) under thefollowing conditions: 95 °C for 5 min, 30 cycles (95 °C for 30 s, 60 °Cfor 30 s and 72 °C for 1 min) and a final elongation step of 72 °C for8 min. The PCR-products were purified using a High Pure PCR ProductPurification Kit (Roche Applied Science, Penzberg, Germany) accord-ing to the manufacturer's instructions. Sequencing was performedwith the amplification primers using a 3130 XL Genetic Analyzer(Applied Biosystems, Foster City, CA, USA) using the ABI Prism Big DyeTerminator Cycle Sequencing Ready Reaction Kit (PE AppliedBiosystems, Nieuwerkerk a/d IJssel, Netherlands). Forward andreverse strands of the aprX gene were assembled with Kodon 3.6software (Applied Maths, Sint-Martens-Latem, Belgium) and alignedwith sequences retrieved from the EMBL database using ClustalX(Thompson et al., 1994). A phylogenetic tree based on aprX genesequences was constructed using Treecon software (Van de Peer andDe Wachter, 1994), applying the neighbour joining algorithmwithoutcorrections. Statistical evaluation of the tree topology was performedby bootstrap analysis with 1000 resamplings. Newly determinedsequences were deposited in the EMBL database (accession numbersare listed in Table 1).

2.5. Specificity and sensitivity of the aprX-PCR towards P. fragi andP. fluorescens in milk

To estimate the detection limit of the developed PCR, P. fluorescensand P. fragi were grown separately in 10% (w/v) reconstituted skimmilk. Each culture was serially diluted in reconstituted skim milkin order to obtain counts ranging from 10 to 108 cfu/ml. PCR speci-ficity towards P. fragi and P. fluorescens was also evaluated in thepresence of other bacterial contaminants in reconstituted skim milk.Therefore a mix of Bacillus cereus, Escherichia coli, Serratia marcescensand Acinetobacter baumanii was grown 24 h at 22 °C in reconstitutedmilk together with a P. fragi or P. fluorescens strain. The mix withoutPseudomonas strains was used as a negative control. To compare thesensitivity of the PCR assay in the presence of other species with the

Fig. 1. Detection of native proteases by SDS-PAGE casein zymogram analysis. Milk samples wafter 48 h of growth in UHT-milk during 2 days at 22 °C. The circle indicates the clearance zobetween the intensity of the clearance zone (protease activity) and the intensity of the mil

detection limit of pure Pseudomonas strains, a dilution PCR wasperformed on milk samples spiked with approximately 106 cfu/mL ofthe bacterial mix together with Pseudomonas strains ranging from 103

to 105 cfu/mL. Total bacterial counts in the milk samples wereevaluated. To discriminate for Pseudomonas strains, all milk sampleswere also evaluated on CFC supplemented Pseudomonas agar (Oxoid).Extraction of bacterial DNA from the milk samples was performedusing a commercially available DNA extraction kit (Adiapure®,Adiagene, France) according to the manufacturer's instructions. In afinalizing step, the DNA extract was washed with chloroform (1:1)(Sigma, Bornem, Belgium) and centrifuged for 10 min at 14,000 ×g toremove impurities. The PCR reaction was performed as describedabove.

2.6. Protein analysis by electrophoresis and mass spectrometry

Pseudomonas strains were grown in full fat UHT-milk at 7 °C for14 days. Subsequently, 10 ml milk culture was adjusted to pH 4.6 byadding HCl (2 M) to precipitate caseins. Samples were centrifuged for5 min at 6500 g. 1 ml supernatant was collected in a fresh tube and100% (w/v) trichloroacetic acid (TCA) was added to precipitate theproteins. Samples were centrifuged for 5 min at 13,000 ×g. Super-natant was removed and the pellet was subsequently washed withethanol, ethanol/diethylether (1:1) and diethylether. The pellet wasdried and resuspended in sample buffer (62.5 mM Tris, 25% (v/v)glycerol, 0.01% (v/v) bromophenol blue, 4% (w/v) Sodium DodecylSulfate (SDS), pH 6.8). 40 μl was loaded onto a 12% Tris–HCl Citerion™precast gel (BioRad). The samples were run at 150 V for 60 min. Afterelectrophoresis, the gel was slightly stained with 0.1% CoomassieBrilliant Blue dissolved in a mixture of 40% ethanol (v/v) and 10%acetic acid (v/v). The bands of interest (as determined by zymogramanalysis) were excised carefully and washed (3×15 min) with200 mM ammonium hydrogen carbonate in 50% (v/v) acetonitrile(ACN), dried in vacuum and re-hydrated in 50 mM ammoniumhydrogen carbonate containing 2 ng/µl modified trypsin (Promega,Leiden, Netherlands). After overnight incubation at 37 °C, thesupernatants were recovered. The gels slices were extracted twicewith 60% (v/v) ACN containing 0.1% (v/v) formic acid. The super-natants were collected again, pooled and dried in vacuum. Theextracted peptides were then diluted with 15 μl 0.1% (v/v) formic acid.

ere heated as described in Materials and methods and diluted 33 times in sample buffernes of casein hydrolysis. The zymogram also clearly illustrates the inverse relationshipk casein protein band (±20 kDa).


Mass spectrometric analyses were carried out on a 4700 or 4800 massspectrometer (Applied Biosystems, Foster City, CA, USA).1 μl of peptidesolutionwasmixedwith equal volume of 10mg/ml HCCA (a-cyano-4-hydroxycinnamic acid) in 50% (v/v) ACN containing 0.1% (v/v)trifluoroacetic acid (TFA), and 1 μl was deposit on the target plate.For MALDI MS/MS analyses, the extracted peptides were desaltedwith C18 Zip tips (Millipore, Brussels, Belgium) and eluted with 5 μl1% formic acid in 80% (v/v) ACN. 0.5 μl of peptide solution wasmixed with equal volume of 5 mg/ml HCCA in 50% (v/v) ACN con-taining 0.1% (v/v) TFA, and then deposited on the target plate. Iden-tification of proteins from MS and/or MS/MS MALDI data wasperformed using the MASCOT algorithm (Matrixscience, Boston, MA,USA) against the non-redundant NCBI database. The MS/MS data of

Fig. 2. aprX-based phylogenetic analysis. Rooted neighbour-joining tree was based on partivalues were generated from 1000 replicates of neighbour joining and values higher than 70

unidentified peptides were examined manually to verify if the frag-mentation patterns matched with those of known Pseudomonasproteases.

3. Results

3.1. Protease production

3.1.1. Casein zymography of milk Pseudomonas strains grown at 22 °CThe 29 Pseudomonas milk strains (of which 9 P. fragi, 5 P. lundensis

and 15 members of the P. fluorescens group) were incubated in full fatUHT-milk for 2 days at 22 °C and their heat-resistant proteolyticactivity was evaluated on casein zymography (Fig. 1). All Pseudomonas

al aprX sequences (850 bp) with Pseudomonas aeruginosa PAO1 as outgroup. Bootstrap% are given. Strains in bold represent milk isolates.


strains produced one protease of similar molecular weight rangingfrom 39.2±0.7 to 45.3±1.3 kDa after 2 days of incubation in full fatUHT-milk at room temperature. When the same strains wereevaluated after 8 days of incubation, four strains (Z34b, W30a, W17cand W12b) displayed 2 or 3 clearance zones of lower molecularweight on casein zymography (data not shown).

3.1.2. Evaluation of protease production at 7 °C, 22 °C and 30 °C of milkPseudomonas strains and reference strains

Growth was visible for all Pseudomonas milk strains at all tem-peratures. As expected, all milk isolates displayed proteolytic activityafter 10 days of incubation at 7 °C and at 22 °C on milk agar plates.Similar results were obtained at 30 °C, except for the P. fragi milkstrains, which failed to produce proteases at this temperature. AllPseudomonas reference strains included in this study showed normalgrowth patterns at all temperatures, except Pseudomonas luteola LMG7041T, which was unable to grow at 7 °C. Despite normal growthpatterns, only 10 out of 26, 13 out of 26 and 13 out of 26 of thePseudomonas reference strains displayed proteolytic activity at 30 °C,22 °C and 7 °C, respectively. The remaining non-Pseudomonas ref-erence strains displayed growth at 30 °C and 22 °C, but the majority(7 out of 9) was unable to growat 7 °C.Within this strain set, 5 out of 9produced proteases at 30 °C and 22 °C, but none showed proteaseproduction at 7 °C. Individual results are listed in Table 1.

3.2. Development of an aprX screening PCR: amplification and sequencingof the aprX gene in Pseudomonas milk strains and reference strains

PCR reactions were performedwith the different primer couples asdescribed in Materials and methods. Based on these preliminaryamplification results, primer set SM2F/SM3R (with an amplificationproduct of approximately 800 bp) was chosen to study the presence ofthe aprX gene in a set of 64 possible contaminants of raw milk amongwhich twenty-nine Pseudomonas milk strains and thirty-five repre-sentatives of potential milk spoiling bacteria available in public culturecollections. All milk strains belonged to the genus Pseudomonas,mainly to P. fragi and P. lundensis. Table 1 provides an overview of alltested strains and their amplification results. Forty-two out of 55Pseudomonas (milk and reference) strains rendered an amplicon ofthe expected size (±850 bp) with the primer set used. The aprX genecould not be demonstrated in the P. lundensis milk strains (W52b,

Fig. 3. Evaluation of the specificity and sensitivity of the aprX-PCR. The detection limit of P. frdilutions (counts in cfu/ml) were loaded into lanes 1–8: 2.2×108 (1), 2.1×107 (2), 1.1×106 (control (10) and P. fluorescens dilutions were loaded into lanes 11–18: 1.2×108 (11), 9.5×10(18), blanc (19). To evaluate the effect of other bacteria on PCR specificity (C), a mix of Bacilluat 22 °C in reconstituted milk powder in the absence (lane 23) or in the presence of 2.2×1

W22b, Z60a, Z24a, Z35c) and type strain (LMG 13517T), P. fragi milkstrain W12d, Pseudomonas milk strain W5a, P. marginalis LMG 14572,P. syringae LMG 1247T, P. aeruginosa LMG 1242T, P. luteola LMG 7041T

and P. psychrophila LMG 24276T, nor in any of the non-Pseudomonasreference strains included. In addition, Table 1 revealed that the aprXgene could still be demonstrated in strains regardless of their proteaseproduction capacity at the investigated temperatures (e.g. P. fragi LMG2191T, LMG 5919 and LMG 5920 display no proteolytic activity at 30,22 °C and 7 °C, yet the aprX gene could still be demonstrated). Allindividual results are listed in Table 1. Sequence similarity analysis anda blast search of the sequenced DNA fragments confirmed that all PCRproducts were indeed aprX fragments.

3.3. Assessment of aprX gene heterogeneity in milk Pseudomonas spp.

The heterogeneity of the protease gene was evaluated by se-quencing all obtained amplicons of ±800 bp. In a phylogenetic clus-tering (Fig. 2) based on partial aprX sequences, 20 Pseudomonas milkisolates grouped together in 4 distinct clusters (A-D) supported byhigh bootstrap values. Three milk isolates (Z34a, Z38b and W51e)formed a distinct cluster (A) with 4 reference P. fluorescens biotype Cstrains. Surprisingly, the aprX gene sequence of the P. gessardii typestrainwas also present in this cluster. The second cluster (B) consistedof solely 5 milk Pseudomonas strains. (W31b, W38a, W12b, W15a andZ57b). The aprX gene sequences of these strains did not match withany of the included type strains, which is indicative for geneticheterogeneity of this protease that might coincide with a new species.Four other milk isolates (Z34b, Z22b, W30a and W2a) clusteredtogether in a rather heterogeneous group (cluster C), which could bedivided in two subclusters (C1 and C2), although not supported by ahigh bootstrap value. Subcluster C1 represents a rather homogenousgroup containing milk strain P. fluorescens Z34b, the P. fluorescens(biotype A) type strain, two other P. fluorescens biotype A representa-tives and the P. tolaasii type strain. Subcluster C2 harbours three milkisolates (Z22b,W30a andW2a), P. rhodesiae LMG 17764T, P. fluorescensbiotype G LMG 5940, P. azotoformans LMG 21611T and P. fluorescensLMG 6812. Cluster D represents the P. fragi group, containing theP. fragi type strain, two other P. fragi reference strains and eight P. fragimilk isolates. The two remaining Pseudomonas milk isolates (W17aand W17c) did not cluster significantly with any reference strainincluded.

agi (A) and P. fluorescens (B) was determined in reconstituted skim milk powder. P. fragi3), 1.1×105 (4), 5.0×103 (5), 2.7×103 (6), 9.6×102 (7), 5.2×102 (8), blanc (9), positive6 (12), 8.7×105 (13), 1.6×105 (14), 1.0×104 (15), 6.3×103 (16), 4.0×103 (17), 2.1×103

s cereus, Escherichia coli, Serratia marcescens and Acinetobacter baumaniiwas grown 24 h07 cfu/ml P. fragi strains (lane 21) or 9.0×106 cfu/ml P. fluorescens strain (lane 22).


The tree topology displayed a rather high level of heterogeneity inthe protease genes of the Pseudomonas milk isolates. The aprX genesequence similarity ranged from 75% to 99% and from 78% to 99% formilk isolates belonging to the P. fluorescens group (clusters A–C) andP. fragi group (cluster D), respectively. Heterogeneity became evenmore distinct, with similarity values ranging from 63% to 72%, whensequences of both groupswere compared to each other. Similar resultswere obtained when the deduced amino acid sequences were com-pared (data not shown). These similarity values illustrate a slightlycloser relatedness of proteases within the above mentioned groups(ranging from 76% to 99% and from 84% to 99% for the P. fluorescensand P. fragi group, respectively). On the contrary, heterogeneitybetween these groups appeared to be more pronounced at the amino

Fig. 4. Multiple sequence alignment of deduced amino acid sequences of the partial aprX gePeptides as retrieved by mass spectrometry are underlined and shown in bold. The three pepin bold in the corresponding region of the protein. Symbols (.), (:) and (⁎) represent the de

acid level than at the nucleotide level with similarity values rangingfrom 57% to 69% (data not shown).

3.4. Specificity and sensitivity of the aprX-PCR test towards P. fragi andP. fluorescens in reconstituted skim milk samples

The amplified aprX-product could be easily detected in reconsti-tutedmilk inoculated with 2.7×103 to 2.2×108 cfu/ml and 2.1×103 to1.2×108 cfu/ml for P. fragi (Fig. 3A) and P. fluorescens (Fig. 3B),respectively. PCR specificity towards P. fragi and P. fluorescens in thepresence of other bacterial contaminants in reconstituted skimmilk isillustrated in Fig. 3C. Total bacterial counts in reconstituted milkreached 4.3×108 cfu/ml for the bacterial mix containing P. fragi (lane

ne obtained for representatives of the four major clusters in the neighbour-joining tree.tides obtained from the Pseudomonas lundensis protease are also underlined and showngree of conservation within the compared sequences.


21) as well as for P. fluorescens (lane 22) and 3.8×108 cfu/ml for thecontrol mix without Pseudomonas strains. Pseudomonas countsreached 2.2×107 (lane 21) and 9.0×106 (lane 22) cfu/ml on CFCagar for the P. fragi and P. fluorescens containing mix, respectively.Fig. 3C illustrates that the aprX products are still detectable in milk inthe presence of other bacterial contaminants (Lane 21 and 22). In thecontrol mix, no amplification products were obtained (Lane 23).Although the specificity of the assay is maintained in the presence ofother bacteria, the dilution PCR illustrated a slight reduction in sen-sitivity. For P. fluorescens as well as P. fragi, detection was visible until104 cfu/ml in a 106 cfu/ml excess of other bacterial contaminants(data not shown). However, pseudomonads dominate the raw milkmicrobiota in a real setting. Thus the observed reduction in sensitivitywill almost be irrelevant when used for the screening of farm milksamples.

3.5. Confirmation of protease identity by mass spectrometry

The produced proteases as visualized on the zymogram in Fig. 1were analyzed by mass spectrometry to verify if they correspond atthe amino acid level to the obtained aprX gene sequences. Fig. 4 showsthe aligned deduced amino acid sequences of 7 representatives of thefour major clusters in the aprX-based phylogenetic tree shown inFig. 2. As can be seen, the retrieved peptides – as obtained by massspectrometry (underlined and shown in bold in Fig. 4) – correspondedperfectly to the individual deduced amino acid sequences. Thus, theactive proteases are indeed the products of the aprX genes. Inaddition, mass spectrometrywas used to explore the protein sequenceof the P. lundensis Z24a protease. Unfortunately, only three protease-related peptide sequences were detected. These are shown in Fig. 4.Degenerate primers based on two of these partial amino acidsequences were designed in an attempt to amplify a part of the aprXgene of this strain (see Table 1 in the Appendix), but no proteasespecific amplification product could be detected, evenwith a variety ofPCR settings applied (data not shown). These results complement theearlier findings on heterogeneity of proteases produced by the milkspoiling Pseudomonas population present in raw milk.

4. Discussion

The goal of this study was to explore the presence and possibleconservation of the aprX gene, encoding a heat-resistant metallopro-tease in P. fragi, P. lundensis and members of the P. fluorescens clade,which are the predominant psychrotrophic species isolated fromBelgian raw milk (Marchand et al., 2009). Psychrotrophic Pseudomo-nas spp. are known for their production of metalloproteases in thecold chain of raw milk (Craven and Macauley, 1992; Sorhaug andStepaniak, 1997; Marchand et al., 2009). Those proteases are heat-resistant and can survive UHT processing which results in the spoilageof the processed dairy product upon extended storage. Evidently,characterization of those proteases is absolutely necessary because itenables detection and the set-up of early warning systems, which onits turn would allow a better shelf life assessment by the manufac-turer. Although some Pseudomonas proteases (in particular from P.fluorescens and P. aeruginosa) have been extensively described in thepast (Okuda et al., 1990; Duong et al., 1992; Miyatake et al., 1995; Kimet al., 1997), the occurrence and diversity of proteases produced by themilk spoiling Pseudomonas population has not been addressed.Biochemical analysis using casein zymography revealed that all milkisolates produced one heat-resistant protease (after two days ofincubation in milk at 22 °C) of a similar molecular weight, rangingfrom 39.2 to 45.3 kDa, which is in agreement with reported values ofother researchers (Noreau and Drapeau, 1979; Kohlmann et al., 1991;Ching-hsing and McCallus, 1998; Koka and Weimer, 2000; Nicodèmeet al., 2005; Dufour et al., 2008). Surprisingly, when the same strainswere evaluated after 8 days of incubation in milk, four strains (Z34b,

W30a, W17c and W12b) displayed 2 or 3 clearance zones of lowermolecular weight on casein zymography. This appearance of activelower molecular weight fragments was also reported by Rajmohan etal. (2002) after ultrafiltration of the culture supernatans of a P.fluorescens strain. The appearance of those fragments was attributedto the concentrating effect of the applied ultrafiltration process beforezymogram analysis (Rajmohan et al., 2002). Nevertheless, we assumethat the appearance could be more likely attributed to substratedepletion, by which the protease, being the only protein source left, isdegraded into smaller active fragments.

All tested milk strains produced proteases at 22 °C, but the in-tensity of the protease patterns on zymogram varied considerably.Thismight be indicative for a variable expression and/or activity of theproteases. Besides, this variability in regulation might be temperaturedependent (McKellar and Cholette, 1987; Burger et al., 2000;Nicodème et al., 2005): while 22 °C, which was the incubationtemperature before zymogram analysis, might be an optimal produc-tion temperature for one species, it might be the limiting factor foranother. As observed in the protease activity experiments, all P. fragimilk strains were incapable of producing proteases at 30 °C. Never-theless, extreme high levels of heat-resistant protease productionwere assessed in refrigerated (7 °C) milk by the same P. fragi milkstrains (Marchand et al., 2009).

Since zymogram analysis revealed that all Pseudomonas milkstrains produced proteases of a similarmolecular weight, the presenceand the possible conservation of the aprX gene, which is responsiblefor the production of heat-resistant proteases (at least in P. fluorescens(Burger et al., 2000; Woods et al., 2001; Dufour et al., 2008)), wasexplored. Martins et al. (2005) already illustrated the presence of theaprX gene in proteolytic psychrotrophic bacteria isolated from rawmilk. However, only a small part (194 bp) of the aprX gene wasamplified in their study, very lowsensitivitieswere obtaineddirectly inmilk and it is not obvious if all potential milk spoilers can be detectedwith the proposed method. Therefore a novel aprX-screening PCR testwas developed. An amplicon of 800 bpwas detected in 22 out of the 29milk spoiling and in 20 out of 26 reference Pseudomonas strains tested.In addition, the PCR appeared to be Pseudomonas specific and moresensitive as Pseudomonas counts as low as 103 and 104 cfu/ml weredetectable in pure milk cultures and in milk with an excess of bacterialcontaminants (N106 cfu/mL), respectively. Since the experimentalsettings illustrated detection of Pseudomonas counts until 104 cfu/mLin milk containing a 10 fold higher bacterial load than the authorizedlimit in the EU directive (105 cfu/mL (Directive 92/46EEC)), it can beconcluded that in the real and legal situation, the detection limit willrather approach the sensitivity of the pure Pseudomonas milk culture.However, since this directive is only valid for bulk tank milk collectedon the farm, it is to be expected that in some situations this limit isexceeded due to prolonged refrigerated storage in the dairy plantbefore processing. In such a case proteases may be present in dairyproducts (e.g. UHT milk) – produced from rawmilk which met the EUdirective – and cause taste deterioration and eventually spoilage of theprocessed dairy product before the end of the shelf life. Unfortunately,not all proteolytic milk isolates (e.g. P. lundensis) were picked upwith the primer set used. Since the few peptides of P. lundensis re-trieved by mass spectrometry still displayed similarity with the otherPseudomonas proteases, heterogeneity in the P. lundensis gene at thelevel of the primer annealing sites might be partially responsible forthis. Because P. lundensis is a rather important milk spoiler (Marchandet al., 2009), future work will address the determination of thecomplete amino acid sequence. Fig. 2 illustrates that the aprX gene israther heterogenouswithin all investigated Pseudomonas strains (withsimilarity values ranging from 63% to 99%). Although this hetero-geneity sets limitations to a general detection method for allproteolytic Pseudomonas strains in milk, it offers great potential forthe development of a multiplex-PCR targeting individual aprX genes.Evidently, such a method would allow discrimination between


different milk Pseudomonas strains and could be useful to determinetheir contamination routes in the cold chain of raw milk. However, ifthe priority is giving advice about milk quality and shelf life, thedevelopment of a protein based detection method, e.g. an immuno-logical assay, might be a more interesting option. After all, moleculardetection of the aprX gene doesn't per se necessitate expression ofprotease. This was also illustrated in Table 1, where the aprX gene wasdemonstrated in protease deficient Pseudomonas reference strains: e.g.P. fragi LMG 2191T, LMG 5919 and LMG 5920. Comparison of aprXsequences of proteolytic and protease deficient P. fragi strains revealedno considerable differences, as illustrated by similarity indices of 90–97%. These data complement the findings of Dufour et al. (2008), whostated that differences in extracellular caseinolytic potential do notresult mainly from differences in aprX sequences but rather from adifferent gene expression and/or regulation. Nevertheless, our datarevealed some important features for the development of an immu-nological assay. Since Pseudomonas proteases are very heterogeneous,each cluster (as obtained in Fig. 2) will need to be represented for thegeneration of antibodies. Finally, antibodies against those proteasesand the P. lundensis protease could allow a general immunologicaldetection of all milk spoiling proteases directly in milk.

Since the aprX-based phylogenetic clustering in Fig. 2 seemed tosupport the biotype classification of P. fluorescens as already describedby Palleroni et al. (Palleroni, 1984) and since the rpoB gene is oftenused as an alternative for 16S rDNA in the classification and iden-tification of Pseudomonas spp., this clustering was compared with anrpoB based phylogenetic clustering of the same strains (See Fig. 1 inthe Appendix). Interestingly, within this set of strains the aprX treeseems more discriminative than the rpoB tree (with the most obviousexample being the Pseudomonas fragi group). This complies with anexpected higher taxonomic resolution, butmore datawill be needed toconfirm this. The complexity of the taxonomic situation of P. fluorescenshas been reported on numerous occasions (Palleroni et al., 1972;Champion et al., 1980; Bossis et al., 2000; Marchand et al., 2009),indicating an important genomic variability within and betweenbiovars of P. fluorescens. Our results, however, show that the aprX genesequences support this biovar classification, at least partly (e.g. biovar I(biotype A) and III (biotype C)). Although the aprX gene could not beamplified, with the primer set used, in all investigated Pseudomonasstrains (e.g. P. aeruginosa), we assume (and in the case of P. aeruginosawe know (Duong et al., 1992)) that this gene will be present in allpseudomonads. Although verification and confirmation of the ob-served aprX based clusters by DNA:DNA hybridizations of Pseudomo-nas strains will be necessary, these observations indicate a potentialuse of the aprX gene as taxonomicmarker, which in the endmight helpresolving the current taxonomic deadlock in the P. fluorescens group.

Acknowledgements

This research was supported by a PhD grant of the Institute forAgricultural and Fisheries Research (ILVO). Wewish to thank Ann Vande Walle, Vera Van Den Mergel, Ann Vanhee and Jessy Claeys for thepractical assistance. We also want to express our gratitude to ourcolleagues of the ILVO-PLANT unit, especially Sabine Van Glabeke, forperforming the sequencing experiments.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ijfoodmicro.2009.04.027.

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heterogeneity of heat-resistant proteases from milk pseudomonas species

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